scholarly journals Serum-free medium and hypoxic preconditioning synergistically enhance the therapeutic effects of mesenchymal stem cells on experimental renal fibrosis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Naoki Ishiuchi ◽  
Ayumu Nakashima ◽  
Shigehiro Doi ◽  
Ryo Kanai ◽  
Satoshi Maeda ◽  
...  
Keyword(s):  
Growth Factor ◽  
Renal Fibrosis ◽  
Transforming Growth Factor ◽  
Hypoxic Preconditioning ◽  
Therapeutic Effects ◽  
Proliferative Capacity ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free

Abstract Background Mesenchymal stem cells (MSCs) repair injured tissue in a paracrine manner. To enhance their therapeutic properties, preconditioning with various factors has been researched. We have previously showed that MSCs cultured in serum-free medium (SF-MSCs) promote their immunosuppressive ability, thereby enhancing their anti-fibrotic effect. Here, we examined whether serum-free medium and hypoxic preconditioning synergistically enhance the therapeutic effects of MSCs on renal fibrosis in rats with ischemia–reperfusion injury (IRI). Methods SF-MSCs were incubated under 1% O2 conditions (hypo-SF-MSCs) or 21% O2 conditions (normo-SF-MSCs) for 24 h before collection. After IRI procedure, hypo-SF-MSCs or normo-SF-MSCs were injected through the abdominal aorta. At 7 or 21 days post-injection, the rats were killed and their kidneys were collected to evaluate inflammation and fibrosis. In in vitro experiments, we investigated whether hypo-SF-MSCs enhanced secretion of anti-fibrotic humoral factors using transforming growth factor (TGF)-β1-stimulated HK-2 cells incubated with conditioned medium from hypo-SF-MSCs or normo-SF-MSCs. Results Normo-SF-MSCs showed attenuation of senescence, which increased their proliferative capacity. Although no significant difference in cellular senescence was found between normo-SF-MSCs and hypo-SF-MSCs, hypo-SF-MSCs further increased their proliferative capacity compared with normo-SF-MSCs. Additionally, administration of hypo-SF-MSCs more strongly ameliorated renal fibrosis than that of normo-SF-MSCs. Moreover, although hypo-SF-MSCs strongly attenuated infiltration of inflammatory cells compared with the control rats, which were treated with PBS, this attenuation was almost equal between normo-SF-MSCs and hypo-SF-MSCs. In vitro experiments revealed that hypo-SF-MSCs more significantly inhibited transforming growth factor (TGF)-β/Smad signaling compared with normo-SF-MSCs. Moreover, hypoxic preconditioning increased hepatocyte growth factor (HGF) secretion even under serum-free conditions, whereas knockdown of HGF in hypo-SF-MSCs attenuated inhibition of TGF-β/Smad signaling. Conclusions These results indicate that administration of ex vivo-expanded, hypoxia-preconditioned SF-MSCs may be a useful cell therapy to prevent renal fibrosis.

Platelet-derived growth factor and multiplication-stimulating activity II, but not multiplication-stimulating activity III-2, stimulate [3H]thymidine and [35S]sulphate incorporation by fetal rat costal cartilage in vitro

1984 ◽  
Vol 103 (2) ◽  
pp. 195-203 ◽  
Author(s):  
D. J. Hill ◽  
R. D. G. Milner
Keyword(s):  
Growth Factor ◽  
Costal Cartilage ◽  
Platelet Derived Growth Factor ◽  
Thymidine Uptake ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Fetal Rat ◽  
Rat Fibroblasts

ABSTRACT The actions of partially purified porcine platelet-derived growth factor (PDGF) and highly purified multiplication-stimulating activity (MSA) II and MSA III-2, which are somatomedins, were investigated on the incorporation of [3H]thymidine and [35S]sulphate by fetal rat costal cartilage in vitro. This was compared with their effects in the presence of 1% fetal calf serum (FCS) on the uptake of thymidine by growth-arrested fetal rat fibroblasts. Platelet-derived growth factor at concentrations of 0·21–21 μg/l enhanced the incorporation of both isotopes by fetal cartilage in the presence of 1% FCS, but had an inconsistent action on thymidine uptake and no significant action on sulphate uptake in serum-free medium. Platelet-derived growth factor promoted thymidine uptake by growth-arrested, isolated fetal rat fibroblasts. Multiplication-stimulating activity II (10–100 μg/l) stimulated the uptake of thymidine and sulphate by fetal cartilage in medium containing 1% FCS but had no consistent action in serum-free medium, although MSA II and PDGF had a synergistic effect on thymidine uptake in the absence of serum. Multiplication-stimulating activity III-2 had no consistent action on thymidine or sulphate incorporation by fetal cartilage in either serum-free or serum-supplemented medium. However, the same preparation of MSA III-2 stimulated the uptake of [3H]thymidine into fetal rat fibroblasts with a half-maximal response at a concentration of 5–10 μg/l. The results identify PDGF as a possible mitogenic agent for fetal rat connective tissues in vitro and show a differential sensitivity of fetal cartilage to MSA peptides. J. Endocr. (1984) 103, 195–203

scholarly journals Electric Field–directed Cell Motility Involves Up-regulated Expression and Asymmetric Redistribution of the Epidermal Growth Factor Receptors and Is Enhanced by Fibronectin and Laminin

1999 ◽  
Vol 10 (4) ◽  
pp. 1259-1276 ◽  
Author(s):  
Min Zhao ◽  
Andrew Dick ◽  
John V. Forrester ◽  
Colin D. McCaig
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Directed Migration ◽  
Directed Cell Migration ◽  
Cell Substrate ◽  
Epidermal Growth

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell–substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell–substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor β and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor β and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.

Megakaryocytes Cultured from Patients with IMF Produced Significantly Higher mRNA but Not Protein Levels of Fibrosing Grown Factors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3607-3607
Author(s):  
Jen-Chin Wang ◽  
Tsong H Chang ◽  
Amit Goldberg ◽  
Allan D. Novetsky ◽  
Steven Lichter ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Growth Factor ◽  
Mrna Levels ◽  
Serum Free Medium ◽  
Free Medium ◽  
Myeloid Metaplasia ◽  
Serum Free ◽  
Protein Levels

Abstract Currently, the prevailing concept concerning the etiology of bone marrow fibrosis in patients with idiopathic myelofibrosis (IMF) is that it results from excessive production of fibrosing growth factors including transforming growth factor beta (TGF-B1), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) from megakaryocytes and monocytes. Since megakaryocytes are difficult to isolate from bone marrow in IMF patients, this concept remains speculative. We obtained megakaryocytes (CD41+ cells) from 10-day in vitro culture of blood CD34+ cells in serum-free medium with thrombopoietin and stem cell factors as described and cultured monocytes from isolating blood CD14+ cells. Then quantitative analyses of fibrosing growth factors at the mRNA and protein levels were obtained. mRNA levels were obtained from real-time RT-PCR technique, and protein levels were obtained from ELISA analysis of the supernatant of CD41+ cells cultured 4 h in serum-free medium. The results showed 1) mRNA levels of TGF-B1, PDGF, and FGF produced by the megakaryocytes were significantly elevated in agnogenic myeloid metaplasia (AMM) compared with those in normal controls (p<0.05). While these growth factors were elevated several-fold in AMM compared with other myeloproliferative disorders (MPD) including essential thrombocythemia and polycythemia vera, they were not statistically significant. 2) mRNA levels of TGF-B1 were higher than levels of PDGF or FGF. 3) The mRNA levels of these growth factors produced from CD14+ cells were not significantly elevated in AMM compared with other MPDs or controls; the AMM mRNA levels were significantly elevated only in some patients. 4) The correlation of mRNA levels of these growth factors with the degree of myelofibrosis in AMM was significant with megakaryocytes (r=0.73) but not with monocytes (r=0.23). 5) ELISA analysis of the growth factors from the cultured megakaryocytes showed that, in most of the patients with AMM and other MPDs and in volunteer controls, the growth factors were undetectable, and only a few patients with AMM had significantly elevated protein levels of these growth factors. We conclude thatin IMF, megakaryocytes but not monocytes are the predominant cells producing fibrosing growth factors, andthe failure of finding increased protein levels of these growth factors in the in vitro system suggest that other factors are necessary to initiate translation of these growth factors in the megakaryoctes, and neutrophil emperipolesis with releasing factors may be important in this process.

Platelet derived growth factor (PDGF) stimulates development of bovine embryos during the fourth cell cycle

Development ◽  
1992 ◽  
Vol 115 (3) ◽  
pp. 821-826 ◽  
Author(s):  
R.C. Larson ◽  
G.G. Ignotz ◽  
W.B. Currie
Keyword(s):  
Cell Cycle ◽  
Growth Factors ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Platelet Derived Growth Factor ◽  
Bovine Embryos ◽  
Serum Free Medium ◽  
Free Medium ◽  
Cell Stage ◽  
Serum Free

In vitro produced, 2-cell bovine embryos were cultured in serum-free medium supplemented with various combinations of growth factors to test the hypothesis that these polypeptide factors are able to signal preimplantation development. The developmental arrest that occurs during the 8-cell stage with typical culture methods might be relieved by a growth factor-dependent mechanism that would stimulate expression of the embryonic genome, thereby mimicking events that occur in vivo in the oviduct during the fourth cell cycle (8- to 16-cell stage). Subsequently, other growth factors might promote compaction and blastulation, processes which normally occur in the uterus. The effects of growth factors on early embryos were evaluated using phase contrast microscopy to monitor progression to the 8-cell stage, completion and duration of the fourth cell cycle, and blastocyst formation. Platelet derived growth factor (PDGF) promoted development beyond the 16-cell stage in 39.1% of the 2-cell embryos examined in all experiments. The duration of the fourth cell cycle among these embryos was approximately 26 hours. During development after the 16-cell stage, PDGF reduced the proportion of embryos bastulating from 12.7% to 5.8%; in contrast, transforming growth factor alpha (TGF alpha), acting during the same developmental time period, increased the proportion of embryos blastulating from 8.6% to 40.6%. These results, using serum-free medium, indicated that PDGF signalled completion of the fourth cell cycle. TGF alpha, and perhaps basic fibroblast growth factor (bFGF), promoted blastulation of 16-cell embryos during subsequent culture.

Effects of insulin-like growth factor-I (IGF-I) and IGF-II on the growth of antler cells in vitro

1994 ◽  
Vol 143 (3) ◽  
pp. 461-469 ◽  
Author(s):  
M Sadighi ◽  
S R Haines ◽  
A Skottner ◽  
A J Harris ◽  
J M Suttie
Keyword(s):  
Growth Factor ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Antler Growth ◽  
Igf I ◽  
And Cartilage

Abstract The effects of insulin-like growth factors -I and -II (IGF-I and -II) on the growth of undifferentiated (fibroblast zone) cells from the growing tip of red deer velvet antlers and from cells 1·5 cm distal to the growing tip (cartilage zone) were investigated in primary cell culture. The addition of IGF-I or IGF-II to the medium of cultures preincubated in serum-free medium for 24 h increased the rate of [3H]thymidine uptake in a dose-dependent manner in both cell types, with maximal stimulation occurring when 1 nm–30 nm was added. The addition of IGF-II to the incubation medium containing IGF-I did not cause a further increase in [3H]thymidine uptake in either cell type over and above each growth factor alone, indicating that there were unlikely to be synergistic effects of IGF-II on the mitogenicity of IGF-I. Binding studies were carried out using 3 × 105 fibroblast zone cells and cartilage zone cells after they had been incubated in serum-free medium for 24 h. 125I-Labelled IGF-I (10−9 m) in a final volume of 200 μl was added to each culture and incubation carried out at 4 °C for a further hour. 125I-Labelled IGF-I bound specifically to both fibroblasts and cartilage zone cells; binding was displaced by both unlabelled IGF-I and by IGF-I antibody. These findings indicate that IGF-I and IGF-II are important mediators for antler growth in vitro and suggest that in view of correlations between IGF-I and antler growth, IGF is functionally significant in controlling velvet antler growth in vivo. Journal of Endocrinology (1994) 143, 461–469

Growth factor-extracellular matrix synergy in the control of trophoblast invasion

2000 ◽  
Vol 28 (2) ◽  
pp. 199-202 ◽  
Author(s):  
J. D. Aplin ◽  
H. Lacey ◽  
T. Haigh ◽  
C. J. P. Jones ◽  
C.-P. Chen ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
First Trimester ◽  
Trophoblast Invasion ◽  
Cell Phenotype ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Igf I

At the periphery of the human placenta, trophoblast attaches to the uterine wall. The tissue interface contains many anchoring sites, with cytotrophoblast columns that form bridges between the overlying extraembryonic (villous) mesenchyme and the maternal decidual stroma beneath. From the periphery of these columns, large numbers of trophoblast cells detach, migrate through the decidua and eventually colonize and transform maternal arteries. In this way the placenta increases and gives priority to the maternal blood supply to the conceptus. We have shown that when early villous tissue is explanted on a collagen gel in serum-free medium, anchoring-site morphogenesis occurs. Thus, in the presence of placental mesenchyme but in the absence of maternal cells, contact with a permissive extracellular matrix (ECM) is necessary and sufficient for cytotrophoblast column development. Proliferation of trophoblast occurs, followed by differentiation into a columnar cell phenotype in which cells remain attached to one another and to the ECM. At this stage, interaction between fibronectin and integrin α5β1 at the cell surface stabilizes the column and the cells remain as a contiguous multilayered sheet. However, the addition of serum-free conditioned medium from first-trimester placental fibroblasts stimulates cytotrophoblast to detach from the distal column and migrate in streams across the ECM. The removal of insulin-like growth factor I (IGF-I) from the fibroblast medium decreases streaming activity, whereas the addition of exogenous IGF-I (10 ng/ml) to serum-free medium produces a streaming phenotype. In contrast, transforming growth factor β1 (10 ng/ml) maintains the cells in a tight sheet. These results suggest the possibility of a paracrine interaction between villous mesenchyme and cytotrophoblast in anchoring sites to stimulate the infiltration of the maternal ECM by trophoblast. Such a mechanism would be self-limiting because the signal diminishes with distance from the placenta.

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